RESUMEN
Understanding of the mechanisms contributing to the increased maternal susceptibility for major adverse cardiovascular events in the postpartum period remains poor. Accordingly, this study tested the hypothesis that the balance between coronary blood flow and myocardial metabolism is compromised during the puerperium period (35-45 days post-delivery) in swine. Systemic and coronary hemodynamic responses were assessed in anesthetized, open-chest control (nonpregnant) and puerperium/postpartum swine at baseline and in response to intravenous infusion of dobutamine (1-30 µg/kg/min). Blood pressure and heart rate were lower in postpartum swine at baseline and in response to dobutamine (P < 0.05). Coronary blood flow and myocardial oxygen delivery were significantly diminished at baseline in postpartum swine (P < 0.001), which corresponded with â¼35% reduction in myocardial oxygen consumption (MVO2) (P < 0.001). Postpartum swine displayed enhanced retrograde coronary flow, larger cardiomyocyte area (P < 0.01) and marked capillary rarefaction (P < 0.01). The relationship between coronary blood flow and heart rate (P < 0.05) or MVO2 (P < 0.001) was significantly diminished in postpartum swine as dobutamine increased MVO2 up to â¼135% in both groups. This reduction in myocardial perfusion was associated with decreases in myocardial lactate uptake (P < 0.001), increases in coronary venous PCO2 (P < 0.01) and decreased coronary venous pH (P < 0.01). These findings suggest an impaired balance between coronary blood flow and myocardial metabolism could contribute to the increased incidence of maternal myocardial ischemia and premature death in the postpartum period.
RESUMEN
The coronary circulation has the inherent ability to maintain myocardial perfusion constant over a wide range of perfusion pressures. The phenomenon of pressure-flow autoregulation is crucial in response to flow-limiting atherosclerotic lesions which diminish coronary driving pressure and increase risk of myocardial ischemia and infarction. Despite well over half a century of devoted research, understanding of the mechanisms responsible for autoregulation remains one of the most fundamental and contested questions in the field today. The purpose of this review is to highlight current knowledge regarding the complex interrelationship between the pathways and mechanisms proposed to dictate the degree of coronary pressure-flow autoregulation. Our group recently likened the intertwined nature of the essential determinants of coronary flow control to the symbolically unsolvable "Gordian knot". To further efforts to unravel the autoregulatory "knot", we consider recent challenges to the local metabolic and myogenic hypotheses and the complicated dynamic structural and functional heterogeneity unique to the heart and coronary circulation. Additional consideration is given to interrogation of putative mediators, role of K+ and Ca2+ channels, and recent insights from computational modeling studies. Improved understanding of how specific vasoactive mediators, pathways, and underlying disease states influence coronary pressure-flow relations stands to significantly reduce morbidity and mortality for what remains the leading cause of death worldwide.
Asunto(s)
Circulación Coronaria , Homeostasis , Humanos , Circulación Coronaria/fisiología , Animales , Presión Sanguínea/fisiología , Vasos Coronarios/fisiopatología , HemodinámicaRESUMEN
The coronary circulation has an innate ability to maintain constant blood flow over a wide range of perfusion pressures. However, the mechanisms responsible for coronary autoregulation remain a fundamental and highly contested question. This study interrogated the local metabolic hypothesis of autoregulation by testing the hypothesis that hypoxemia-induced exaggeration of the metabolic error signal improves the autoregulatory response. Experiments were performed on open-chest anesthetized swine during stepwise changes in coronary perfusion pressure (CPP) from 140 to 40 mmHg under normoxic (n = 15) and hypoxemic (n = 8) conditions, in the absence and presence of dobutamine-induced increases in myocardial oxygen consumption (MVO2) (n = 5-7). Hypoxemia (PaO2 < 40 mmHg) decreased coronary venous PO2 (CvPO2) ~ 30% (P < 0.001) and increased coronary blood flow ~ 100% (P < 0.001), sufficient to maintain myocardial oxygen delivery (P = 0.14) over a wide range of CPPs. Autoregulatory responsiveness during hypoxemia-induced reductions in CvPO2 were associated with increases of autoregulatory gain (Gc; P = 0.033) but not slope (P = 0.585) over a CPP range of 120 to 60 mmHg. Preservation of autoregulatory Gc (P = 0.069) and slope (P = 0.264) was observed during dobutamine administration ± hypoxemia. Reductions in coronary resistance in response to decreases in CPP predominantly occurred below CvPO2 values of ~ 25 mmHg, irrespective of underlying vasomotor reserve. These findings support the presence of an autoregulatory threshold under which oxygen-sensing pathway(s) act to preserve sufficient myocardial oxygen delivery as CPP is reduced during increases in MVO2 and/or reductions in arterial oxygen content.
Asunto(s)
Dobutamina , Oxígeno , Porcinos , Animales , Presión Sanguínea , Dobutamina/farmacología , Miocardio/metabolismo , Circulación Coronaria/fisiología , Homeostasis/fisiología , Consumo de Oxígeno/fisiología , Hipoxia , PerfusiónRESUMEN
The lack of pre-clinical large animal models of heart failure with preserved ejection fraction (HFpEF) remains a growing, yet unmet obstacle to improving understanding of this complex condition. We examined whether chronic cardiometabolic stress in Ossabaw swine, which possess a genetic propensity for obesity and cardiovascular complications, produces an HFpEF-like phenotype. Swine were fed standard chow (lean; n = 13) or an excess calorie, high-fat, high-fructose diet (obese; n = 16) for ~ 18 weeks with lean (n = 5) and obese (n = 8) swine subjected to right ventricular pacing (180 beats/min for ~ 4 weeks) to induce heart failure (HF). Baseline blood pressure, heart rate, LV end-diastolic volume, and ejection fraction were similar between groups. High-rate pacing increased LV end-diastolic pressure from ~ 11 ± 1 mmHg in lean and obese swine to ~ 26 ± 2 mmHg in lean HF and obese HF swine. Regression analyses revealed an upward shift in LV diastolic pressure vs. diastolic volume in paced swine that was associated with an ~ twofold increase in myocardial fibrosis and an ~ 50% reduction in myocardial capillary density. Hemodynamic responses to graded hemorrhage revealed an ~ 40% decrease in the chronotropic response to reductions in blood pressure in lean HF and obese HF swine without appreciable changes in myocardial oxygen delivery or transmural perfusion. These findings support that high-rate ventricular pacing of lean and obese Ossabaw swine initiates underlying cardiac remodeling accompanied by elevated LV filling pressures with normal ejection fraction. This distinct pre-clinical tool provides a unique platform for further mechanistic and therapeutic studies of this highly complex syndrome.
Asunto(s)
Insuficiencia Cardíaca , Animales , Fructosa , Obesidad/complicaciones , Oxígeno , Fenotipo , Volumen Sistólico/fisiología , Porcinos , Función Ventricular IzquierdaRESUMEN
Impaired coronary microvascular function (e.g., reduced dilation and coronary flow reserve) predicts cardiac mortality in obesity, yet underlying mechanisms and potential therapeutic strategies remain poorly understood. Mineralocorticoid receptor (MR) antagonism improves coronary microvascular function in obese humans and animals. Whether MR blockade improves in vivo regulation of coronary flow, a process involving voltage-dependent K+ (Kv) channel activation, or reduces coronary structural remodeling in obesity is unclear. Thus, the goals of this investigation were to determine the effects of obesity on coronary responsiveness to reductions in arterial PO2 and potential involvement of Kv channels and whether the benefit of MR blockade involves improved coronary Kv function or altered passive structural properties of the coronary microcirculation. Hypoxemia increased coronary blood flow similarly in lean and obese swine; however, baseline coronary vascular resistance was significantly higher in obese swine. Inhibition of Kv channels reduced coronary blood flow and augmented coronary resistance under baseline conditions in lean but not obese swine and had no impact on hypoxemic coronary vasodilation. Chronic MR inhibition in obese swine normalized baseline coronary resistance, did not influence hypoxemic coronary vasodilation, and did not restore coronary Kv function (assessed in vivo, ex vivo, and via patch clamping). Lastly, MR blockade prevented obesity-associated coronary arteriolar stiffening independent of cardiac capillary density and changes in cardiac function. These data indicate that chronic MR inhibition prevents increased coronary resistance in obesity independent of Kv channel function and is associated with mitigation of obesity-mediated coronary arteriolar stiffening.
Asunto(s)
Aldosterona/farmacología , Enfermedad de la Arteria Coronaria/prevención & control , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Antagonistas de Receptores de Mineralocorticoides/farmacología , Obesidad/tratamiento farmacológico , Canales de Potasio con Entrada de Voltaje/metabolismo , Resistencia Vascular/efectos de los fármacos , Animales , Arteriolas/efectos de los fármacos , Arteriolas/metabolismo , Arteriolas/fisiopatología , Enfermedad de la Arteria Coronaria/etiología , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/fisiopatología , Vasos Coronarios/metabolismo , Vasos Coronarios/fisiopatología , Modelos Animales de Enfermedad , Femenino , Masculino , Microcirculación/efectos de los fármacos , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/fisiopatología , Sus scrofa , Rigidez Vascular/efectos de los fármacosRESUMEN
The "two-hit vascular hypothesis for Alzheimer's disease (AD)" and amyloid-ß (Aß) oligomer hypothesis suggest that impaired soluble Aß oligomers clearance through the cerebral vasculature may be an initial step of the AD process. Soluble Aß oligomers are driven into perivascular spaces from the brain parenchyma and toward peripheral blood flow. The underlying vascular-based mechanism, however, has not been defined. Given that microRNAs (miRNAs), emerging as novel modulators, are involved in numerous physiological and pathological processes, we hypothesized that cerebrovascular miRNAs may regulate the activities of brain blood vessels, which further affects the concentration of Aß in the AD brain. In this study, perivascular Aß deposits, higher vascular activation, increased pericyte coverage and up-regulated capillaries miRNAs at 6 months old (6 mo) were found to correlate with the lower Aß levels of middle AD stage (9 mo) in 3xTg-AD (3xTg) mice. It is implicated that at the early stage of AD when intracellular Aß appeared, higher expression of vessel-specific miRNAs, elevated pericyte coverage, and activated endothelium facilitate Aß oligomer clearance through the perivascular route, resulting in a transient reduction of Aß oligomers at 9 mo. Additionally, ghrelin-induced upregulation of capillary miRNAs and increased pericyte coverage attenuated Aß burden at 9 mo, in further support of the relationship between vascular miRNAs and Aß clearance. This work suggests a cerebral microvessel miRNA may boost endothelial highly activated phenotypes to promote elimination of Aß oligomers through the perivascular drainage pathway and contribute to AD progression. The targeting of brain vessel-specific miRNAs may provide a new rationale for the development of innovative therapeutic strategies for AD treatment.
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Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/patología , Capilares/metabolismo , Circulación Cerebrovascular/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
The local metabolic hypothesis proposes that myocardial oxygen tension determines the degree of autoregulation by increasing the production of vasodilator metabolites as perfusion pressure is reduced. Thus, normal physiologic levels of coronary venous PO2, an index of myocardial oxygenation, are proposed to be required for effective autoregulation. The present study challenged this hypothesis through determination of coronary responses to changes in coronary perfusion pressure (CPP 140-40 mmHg) in open-chest swine in the absence (n = 7) and presence of euvolemic hemodilution (~ 50% reduction in hematocrit), with (n = 5) and without (n = 6) infusion of dobutamine to augment MVO2. Coronary venous PO2 decreased over similar ranges (~ 28-15 mmHg) as CPP was lowered from 140 to 40 mmHg in each of the groups. However, coronary venous PO2 was not associated with changes in coronary blood flow (r = - 0.11; P = 0.29) or autoregulatory gain (r = - 0.29; P = 0.12). Coronary zero-flow pressure (Pzf) was measured in 20 mmHg increments and determined to be directly related to vascular resistance (r = 0.71; P < 0.001). Further analysis demonstrated that changes in coronary blood flow remained minimal at Pzf > 20 mmHg, but progressively increased as Pzf decreased below this threshold value (r = 0.68; P < 0.001). Coronary Pzf was also positively correlated with autoregulatory gain (r = 0.43; P = 0.001). These findings support that coronary autoregulatory behavior is predominantly dependent on an adequate degree of underlying vasomotor tone, independent of normal myocardial oxygen tension.
Asunto(s)
Circulación Coronaria , Vasos Coronarios/metabolismo , Hemodinámica , Miocardio/metabolismo , Oxígeno/sangre , Sistema Vasomotor/metabolismo , Agonistas de Receptores Adrenérgicos beta 1/farmacología , Animales , Presión Sanguínea , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Dobutamina/farmacología , Hemodilución , Homeostasis , Masculino , Modelos Animales , Transducción de Señal , Sus scrofa , Vasodilatación , Sistema Vasomotor/fisiologíaRESUMEN
Myogenic responses (pressure-dependent contractions) of coronary arterioles play a role in autoregulation (relatively constant flow vs. pressure). Publications on myogenic reactivity in swine coronaries vary in caliber, analysis, and degree of responsiveness. Further, data on myogenic responses and autoregulation in swine have not been completely compiled, compared, and modeled. Thus, it has been difficult to understand these physiological phenomena. Our purpose was to: (a) analyze myogenic data with standard criteria; (b) assign results to diameter categories defined by morphometry; and (c) use our novel multiscale flow model to determine the extent to which ex vivo myogenic reactivity can explain autoregulation in vivo. When myogenic responses from the literature are an input for our model, the predicted coronary autoregulation approaches in vivo observations. More complete and appropriate data are now available to investigate the regulation of coronary blood flow in swine, a highly relevant model for human physiology and disease.
RESUMEN
The heart is uniquely responsible for providing its own blood supply through the coronary circulation. Regulation of coronary blood flow is quite complex and, after over 100 years of dedicated research, is understood to be dictated through multiple mechanisms that include extravascular compressive forces (tissue pressure), coronary perfusion pressure, myogenic, local metabolic, endothelial as well as neural and hormonal influences. While each of these determinants can have profound influence over myocardial perfusion, largely through effects on end-effector ion channels, these mechanisms collectively modulate coronary vascular resistance and act to ensure that the myocardial requirements for oxygen and substrates are adequately provided by the coronary circulation. The purpose of this series of Comprehensive Physiology is to highlight current knowledge regarding the physiologic regulation of coronary blood flow, with emphasis on functional anatomy and the interplay between the physical and biological determinants of myocardial oxygen delivery. © 2017 American Physiological Society. Compr Physiol 7:321-382, 2017.
Asunto(s)
Circulación Coronaria/fisiología , Homeostasis/fisiología , Vasos Coronarios/anatomía & histología , Vasos Coronarios/inervación , Endotelio Vascular/fisiología , Hormonas/fisiología , Humanos , Canales Iónicos/fisiología , Miocardio/metabolismo , Consumo de Oxígeno/fisiología , Resistencia Vascular/fisiología , Vasodilatación/fisiologíaRESUMEN
Ion channels in smooth muscle control coronary vascular tone, but the identity of the potassium channels involved requires further investigation. The purpose of this study was to evaluate the functional role of KV1 channels on porcine coronary blood flow using the selective antagonist correolide. KV1 channel gene transcripts were found in porcine coronary arteries, with KCNA5 (encoding KV1.5) being most abundant (P < 0.001). Immunohistochemical staining demonstrated KV1.5 protein in the vascular smooth muscle layer of both porcine and human coronary arteries, including microvessels. Whole-cell patch-clamp experiments demonstrated significant correolide-sensitive (1-10 µM) current in coronary smooth muscle. In vivo studies included direct intracoronary infusion of vehicle or correolide into a pressure-clamped left anterior descending artery of healthy swine (n = 5 in each group) with simultaneous measurement of coronary blood flow. Intracoronary correolide (~0.3-3 µM targeted plasma concentration) had no effect on heart rate or systemic pressure, but reduced coronary blood flow in a dose-dependent manner (P < 0.05). Dobutamine (0.3-10 µg/kg/min) elicited coronary metabolic vasodilation and intracoronary correolide (3 µM) significantly reduced coronary blood flow at any given level of myocardial oxygen consumption (P < 0.001). Coronary artery occlusions (15 s) elicited reactive hyperemia and correolide (3 µM) reduced the flow volume repayment by approximately 30 % (P < 0.05). Taken together, these data support a major role for KV1 channels in modulating baseline coronary vascular tone and, perhaps, vasodilation in response to increased metabolism and transient ischemia.
Asunto(s)
Circulación Coronaria/fisiología , Vasos Coronarios/metabolismo , Músculo Liso Vascular/metabolismo , Canales de Potasio de la Superfamilia Shaker/metabolismo , Animales , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , PorcinosRESUMEN
We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.
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Circulación Coronaria , Angiopatías Diabéticas/metabolismo , Peróxido de Hidrógeno/metabolismo , Microcirculación , Canales Catiónicos TRPV/metabolismo , Animales , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Hydrogen peroxide (H2O2) and voltage-dependent K(+) (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli.
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Circulación Coronaria/fisiología , Vasos Coronarios/metabolismo , Canales de Potasio KCNQ/genética , Comunicación Paracrina/fisiología , Adventicia/metabolismo , Aminopiridinas/farmacología , Animales , Western Blotting , Bradiquinina/farmacología , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/metabolismo , Indoles/farmacología , Canales de Potasio KCNQ/metabolismo , Canal de Potasio KCNQ1/genética , Canal de Potasio KCNQ2/genética , Canal de Potasio KCNQ3/genética , Bloqueadores de los Canales de Potasio/farmacología , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Porcinos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Enhanced sensitivity in echocardiographic analyses may allow for early detection of changes in cardiac function beyond the detection limits of conventional echocardiographic analyses, particularly in a small animal model. The goal of this study was to compare conventional echocardiographic measurements and speckle-tracking based strain imaging analyses in a small animal model of type 1 diabetes mellitus. Conventional analyses revealed differences in ejection fraction, fractional shortening, cardiac output, and stroke volume in diabetic animals relative to controls at 6-weeks post-diabetic onset. In contrast, when assessing short- and long-axis speckle-tracking based strain analyses, diabetic mice showed changes in average systolic radial strain, radial strain rate, radial displacement, and radial velocity, as well as decreased circumferential and longitudinal strain rate, as early as 1-week post-diabetic onset and persisting throughout the diabetic study. Further, we performed regional analyses for the LV and found that the free wall region was affected in both the short- and long-axis when assessing radial dimension parameters. These changes began 1-week post-diabetic onset and remained throughout the progression of the disease. These findings demonstrate the use of speckle-tracking based strain as an approach to elucidate cardiac dysfunction from a global perspective, identifying left ventricular cardiac regions affected during the progression of type 1 diabetes mellitus earlier than contractile changes detected by conventional echocardiographic measurements.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Cardiomiopatías Diabéticas/diagnóstico , Ecocardiografía/métodos , Ventrículos Cardíacos/fisiopatología , Disfunción Ventricular Izquierda/diagnóstico , Animales , Gasto Cardíaco , Cardiomiopatías Diabéticas/fisiopatología , Diástole , Masculino , Ratones , Volumen Sistólico , Sístole , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
OBJECTIVE: Many types of vascular smooth muscle cells exhibit prominent KDR currents. These KDR currents may be mediated, at least in part, by KV1.5 channels, which are sensitive to inhibition by DPO-1. We tested the hypothesis that DPO-1-sensitive KDR channels regulate the tone and reactivity of resistance-sized vessels from rat brain (MCA) and skeletal muscle (GA). METHODS: Middle cerebral and gracilis arteries were isolated and subjected to three kinds of experimental analysis: (i) western blot/immunocytochemistry; (ii) patch clamp electrophysiology; and (iii) pressure myography. RESULTS: Western blot and immunocytochemistry experiments demonstrated KV1.5 immunoreactivity in arteries and smooth muscle cells isolated from them. Whole-cell patch clamp experiments revealed smooth muscle cells from resistance-sized arteries to possess a KDR current that was blocked by DPO-1. Resistance arteries constricted in response to increasing concentrations of DPO-1. DPO-1 enhanced constrictions to PE and serotonin in gracilis and middle cerebral arteries, respectively. When examining the myogenic response, we found that DPO-1 reduced the diameter at any given pressure. Dilations in response to ACh and SNP were reduced by DPO-1. CONCLUSION: We suggest that KV1.5, a DPO-1-sensitive KDR channel, plays a major role in determining microvascular tone and the response to vasoconstrictors and vasodilators.
Asunto(s)
Encéfalo/irrigación sanguínea , Canal de Potasio Kv1.5/metabolismo , Músculo Esquelético/irrigación sanguínea , Compuestos Organofosforados/farmacología , Resistencia Vascular/efectos de los fármacos , Animales , Masculino , Ratones , Ratones Noqueados , Arteria Cerebral Media/metabolismo , Ratas , Ratas Sprague-Dawley , Vasodilatación/efectos de los fármacosRESUMEN
Na-K-ATPase, an integral membrane protein in mammalian cells, is responsible for maintaining the favorable intracellular Na gradient necessary to promote Na-coupled solute cotransport processes [e.g., Na-glucose cotransport (SGLT1)]. Inhibition of brush border membrane (BBM) SGLT1 is, at least in part, due to the diminished Na-K-ATPase in villus cells from chronically inflamed rabbit intestine. The aim of the present study was to determine the effect of Na-K-ATPase inhibition on the two major BBM Na absorptive pathways, specifically Na-glucose cotransport and Na/H exchange (NHE), in intestinal epithelial (IEC-18) cells. Na-K-ATPase was inhibited using 1 mM ouabain or siRNA for Na-K-ATPase-α1 in IEC-18 cells. SGLT1 activity was determined as 3-O-methyl-D-[(3)H]glucose uptake. Na-K-ATPase activity was measured as the amount of inorganic phosphate released. Treatment with ouabain resulted in SGLT1 inhibition at 1 h but stimulation at 24 h. To further characterize this unexpected stimulation of SGLT1, siRNA silencing was utilized to inhibit Na-K-ATPase-α1. SGLT1 activity was significantly upregulated by Na-K-ATPase silencing, while NHE3 activity remained unaltered. Kinetics showed that the mechanism of stimulation of SGLT1 activity was secondary to an increase in affinity of the cotransporter for glucose without a change in the number of cotransporters. Molecular studies demonstrated that the mechanism of stimulation was not secondary to altered BBM SGLT1 protein levels. Chronic and direct silencing of basolateral Na-K-ATPase uniquely regulates BBM Na absorptive pathways in intestinal epithelial cells. Specifically, while BBM NHE3 is unaffected, SGLT1 is stimulated secondary to enhanced affinity of the cotransporter.
Asunto(s)
Absorción Intestinal/fisiología , Microvellosidades/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Transporte Biológico/fisiología , Línea Celular , Coccidiosis/parasitología , Coccidiosis/patología , Eimeria/inmunología , Eimeria/patogenicidad , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/genética , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Intestino Delgado/citología , Masculino , Ouabaína/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Conejos , Ratas , Sodio/metabolismo , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/genética , Intercambiador 3 de Sodio-Hidrógeno , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/genéticaRESUMEN
We demonstrated previously that BK (K(Ca)1.1) channel activity (NP(o)) increases in response to bisphenol A (BPA). Moreover, BK channels containing regulatory ß1 subunits were more sensitive to the stimulatory effect of BPA. How BPA increases BK channel NPo remains mostly unknown. Estradiol activates BK channels by binding to an extracellular site, but neither the existence nor location of a BPA binding site has been demonstrated. We tested the hypothesis that an extracellular binding site is responsible for activation of BK channels by BPA. We synthesized membrane-impermeant BPA-monosulfate (BPA-MS) and used patch clamp electrophysiology to study channels composed of α or α + ß1 subunits in cell-attached (C-A), whole-cell (W-C), and inside-out (I-O) patches. In C-A patches, bath application of BPA-MS (100 µM) had no effect on the NP(o) of BK channels, regardless of their subunit composition. Importantly, however, subsequent addition of membrane-permeant BPA (100 µM) increased the NP(o) of both α and α + ß1 channels in C-A patches. The C-A data indicate that in order to alter BK channel NP(o), BPA must interact with the channel itself (or some closely associated partner) and diffusible messengers are not involved. In W-C patches, 100 µM BPA-MS activated current in cells expressingα subunits, whereas cells expressing α + ß1 subunits responded similarly to a log-order lower concentration (10 µM). The W-C data suggest that an extracellular activation site exists, but do not eliminate the possibility that an intracellular site may also be present. In I-O patches, where the cytoplasmic face was exposed to the bath, BPA-MS had no effect on the NP(o) of BK α subunits, but BPA increased it. BPA-MS increased the NP(o) of α + ß1 channels in I-O patches, but not as much as BPA. We conclude that BPA activates BK α via an extracellular site and that BPA-sensitivity is increased by the ß1 subunit, which may also constitute part of an intracellular binding site.
Asunto(s)
Compuestos de Bencidrilo/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Fenoles/metabolismo , Línea Celular , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Potenciales de la Membrana , Técnicas de Placa-Clamp , Estructura Terciaria de ProteínaRESUMEN
Previous investigations indicate that diminished functional expression of voltage-dependent K(+) (KV) channels impairs control of coronary blood flow in obesity/metabolic syndrome. The goal of this investigation was to test the hypothesis that KV channels are electromechanically coupled to CaV1.2 channels and that coronary microvascular dysfunction in obesity is related to subsequent increases in CaV1.2 channel activity. Initial studies revealed that inhibition of KV channels with 4-aminopyridine (4AP, 0.3 mM) increased intracellular [Ca(2+)], contracted isolated coronary arterioles and decreased coronary reactive hyperemia. These effects were reversed by blockade of CaV1.2 channels. Further studies in chronically instrumented Ossabaw swine showed that inhibition of CaV1.2 channels with nifedipine (10 µg/kg, iv) had no effect on coronary blood flow at rest or during exercise in lean swine. However, inhibition of CaV1.2 channels significantly increased coronary blood flow, conductance, and the balance between coronary flow and metabolism in obese swine (P < 0.05). These changes were associated with a ~50 % increase in inward CaV1.2 current and elevations in expression of the pore-forming subunit (α1c) of CaV1.2 channels in coronary smooth muscle cells from obese swine. Taken together, these findings indicate that electromechanical coupling between KV and CaV1.2 channels is involved in the regulation of coronary vasomotor tone and that increases in CaV1.2 channel activity contribute to coronary microvascular dysfunction in the setting of obesity.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Circulación Coronaria/fisiología , Obesidad/metabolismo , Obesidad/fisiopatología , Canales de Potasio/metabolismo , Animales , Hemodinámica/fisiología , PorcinosRESUMEN
Myocardial metabolites such as adenosine mediate reactive hyperemia, in part, by activating ATP-dependent K(+) (K(ATP)) channels in coronary smooth muscle. In this study, we investigated the role of adenosine A(2A) and A(2B) receptors and their signaling mechanisms in reactive hyperemia. We hypothesized that coronary reactive hyperemia involves A(2A) receptors, hydrogen peroxide (H(2)O(2)), and KATP channels. We used A(2A) and A(2B) knockout (KO) and A(2A/2B) double KO (DKO) mouse hearts for Langendorff experiments. Flow debt for a 15-s occlusion was repaid 128 ± 8% in hearts from wild-type (WT) mice; this was reduced in hearts from A(2A) KO and A(2A)/(2B) DKO mice (98 ± 9 and 105 ± 6%; P < 0.05), but not A(2B) KO mice (123 ± 13%). Patch-clamp experiments demonstrated that adenosine activated glibenclamide-sensitive KATP current in smooth muscle cells from WT and A(2B) KO mice (90 ± 23% of WT) but not A(2A) KO or A(2A)/A(2B) DKO mice (30 ± 4 and 35 ± 8% of WT; P < 0.05). Additionally, H(2)O(2) activated KATP current in smooth muscle cells (358 ± 99%; P < 0.05). Catalase, an enzyme that breaks down H(2)O(2), attenuated adenosine-induced coronary vasodilation, reducing the percent increase in flow from 284 ± 53 to 89 ± 13% (P < 0.05). Catalase reduced the repayment of flow debt in hearts from WT mice (84 ± 9%; P < 0.05) but had no effect on the already diminished repayment in hearts from A(2A) KO mice (98 ± 7%). Our findings suggest that adenosine A(2A) receptors are coupled to smooth muscle KATP channels in reactive hyperemia via the production of H(2)O(2) as a signaling intermediate.
Asunto(s)
Vasos Coronarios/fisiología , Peróxido de Hidrógeno/metabolismo , Hiperemia/fisiopatología , Canales KATP/fisiología , Receptor de Adenosina A2A/fisiología , Transducción de Señal/fisiología , Adenosina/farmacología , Animales , Catalasa/metabolismo , Circulación Coronaria/efectos de los fármacos , Circulación Coronaria/fisiología , Gliburida/farmacología , Hipoglucemiantes/farmacología , Técnicas In Vitro , Canales KATP/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/efectos de los fármacos , NADH NADPH Oxidorreductasas/metabolismo , Técnicas de Placa-Clamp , Receptor de Adenosina A2A/efectos de los fármacos , Receptor de Adenosina A2B/efectos de los fármacos , Receptor de Adenosina A2B/fisiología , Vasodilatadores/farmacologíaRESUMEN
Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.